Many molecular biological methods make use of the capture and solid-phase manipulation of compounds. Such methods include solid-phase DNA sequencing, DNA/RNA hybridisation, separation of polymerase chain reaction (PCR) products, labelling of single-stranded nucleic acid probes, gene assembly, in vitro mutagenesis, yeast artificial chromosome (YAC)-screening, DNA cloning, sequence-specific purification of DNA/RNA binding proteins, cell separation and isolation of bacteria etc.
A number of capture methods rely on the interaction between biotin and biotin-binding proteins such as avidin or streptavidin. Streptavidin is a preferred biotin-binding protein as it has four identical subunits each of which has a high affinity binding site for biotin making it suitable for use in the rapid and efficient isolation of biotin-labelled target molecules. The appropriate biotinylated compounds vary according to the application but include compounds such as double-stranded and single-stranded DNA, RNA, proteins, sugars and lectins.
A popular method for such capture and solid-phase manipulation relies upon the use of streptavidin-coated monodisperse magnetic particles, such as those supplied by Dynal A.S. (Dynabeads™ M-280 Streptavidin) which are highly uniform, superparamagnetic, polystyrene beads coated with a polyurethane layer. The stability of the interaction between biotin and streptavidin enables manipulations such as DNA strand melting, hybridisation and elution to be performed without affecting the immobilisation of the biotinylated compound while the magnetic particles allow separation through exposing the beads to a magnetic field gradient. However, such beads are expensive and cumbersome to produce.
Other methods are known in the art. Thus WO 96/37313 discloses complex magnetically responsive microparticles which can be coated with surface-active molecules, such as streptavidin, to effect separation of biological materials. Similarly, U.S. Pat. No. 5,693,539 describes polymer-coated magnetic particles which may be conjugated to binding moieties such as avidin or biotin for the purification and/or separation of bio-molecules. Biotinylated lipid-coated magnetic nanoclusters bearing avidin surface residues have also been described in the literature for use in affinity capture (Sonti S.& Bose A., Colloids and Surfaces (1997) 8 (4); pp 1999-204). However, as with the DYNABEADS™ M-280 Streptavidin product described above, the preparation of these magnetic particles or nanoclusters is both complex and expensive.
There is therefore a need to provide a simple composition of magnetic particles for the separation and manipulation of biological compounds that can be prepared more readily and more cheaply than the known products.